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Thursday, November 3, 2016

Thinking BIG and small when it comes to DNA cloning

It may seem counterintuitive that a single kit would enable
high-efficiency cloning of both very small (~100 bp) and large (100 kb) DNA fragments,
but the SGI-DNA Gibson Assembly® Ultra kit accomplishes that and more.

Engineered by Daniel Gibson and his team in their pursuit to
synthesize a bacterial genome, the Ultra kit has roots in large-scale synthetic
biology. What Daniel Gibson and other leading scientists have also discovered,
is that the Ultra kit performs exceptionally well in enabling small-scale
routine cloning applications quickly and efficiently as well. The Ultra kit
mediates the seamless cloning of up to 15 DNA fragments in a two-step reaction
in a single tube and can be used with any plasmid or BAC vector. The fragments
for assembly can vary widely in size. Standard assembly of a 100 bp fragment
with a pUC19 vector yields a circular construct less than 3 kb in size. On
the other extreme, multi-stage assembly with the Ultra kit can be used to
engineer large constructs 1,000,000 bp in length (1 Mbp).

Gibson Assembly Ultra Protocol

Completed in an 80-minute reaction with less than 6 minutes
of hands-on time, constructs assembled with the Ultra kit are double-stranded,
fully sealed, and can be used immediately in downstream molecular biology
applications. To perform the Gibson Assembly® Ultra cloning method, prepare DNA
fragments and your vector of choice with overlapping ends. Combine 10–300
ng (in equimolar amounts*) of each DNA fragment, the vector, and Ultra Master
Mix A. After incubating the reaction components through a temperature cycle
enabling chew back and annealing, add Ultra Master Mix B to the reaction.
Following a 15-minute incubation at 45°C, the fully ligated constructs are
ready to transform into competent cells.

*We recommend a 5-fold molar excess
of the insert for DNA fragments.

Like the Gibson Assembly® HiFi 1-Step kit, the Ultra kit is available
as a 5-reaction starter kit. Larger kit sizes, including bulk pricing, are also
available. Whether you are planning a big or a small cloning project, our team
of experts can help. Email us at info@sgidna.com
for assistance.